The present invention is directed to thermally responsive synthetic polymer monoliths. More particularly, the present invention is directed to porous synthetic polymer monoliths wherein the pores contain grafted temperature-responsive polymers and copolymers. Depending on the grafting compositions employed, thermally-responsive monoliths can be produced in which the flow through the micrometer sized pores can be controlled (thermal valves) or completely blocked (thermal gates). When the thermally responsive polymers are in their compact form at elevated temperature, flow through the micrometer sized pores is unimpeded.
"Smart" polymers responsive to changes in pH, temperature, and irradiation have been reported in the literature to have been used in applications such as drug delivery, encapsulated enzyme bio-reactors, and the formation of membranes with controlled permeability. The state-of-the-art in this area has been reviewed recently by Galaev (Russian Chemical Reviews 64, 471-489, 1995).
Galaev discloses using the ability of "smart" polymers to undergo transformation from an uncoiled globule into a compact coil to create membranes with controllable permeability (at 478), it produces structures which exhibit exactly the opposite behavior than exists in the present invention. In Galaev's compact conformation, the "smart" polymer molecules block or strongly hinder the entry of a solute into the pores of the membrane. In the present invention, when the thermally responsive polymer is in its compact form (at high temperature), flow through the monolith is unimpeded.
Galaev also makes reference to a system wherein purification of an enzyme is performed by elution solely as a result of changing the temperature without any alteration of the composition of the buffer (at 482). The system described, however, contains but a single enzyme in a solution and removes that enzyme by adsorption to a "smart" polymer attached to packed beads. The system can not separate two or more enzymes from each other as can the present invention.
U.S. Pat. No. 5,426,154 discloses thermally reversible graft copolymers comprising a vinyl polymer graft copolymerized to a polymer of N-substituted acrylamide or methacrylamide derivatives.
Hydrogel membranes with temperature-controlled permeability for molecules of different sizes (Fief et al., Journal of Membrane Science, 1991, 64, 283) and polarity (T. Ogata et al, Journal of Membrane Science, 1995, 103, 159) were prepared directly by the copolymerization of N-isopropylacrylamide (NIPAAm) with a comonomer and a crosslinking agent. A decrease in the permeability of these membranes was observed at temperatures exceeding the lower critical solution temperature (LCST). This is the opposite of the behavior which occurs in the monoliths of this invention.
NIPAAm has also been grafted onto the surface of commercial polyamide membranes using W irradiation initiated polymerization and the resulting membrane showed a temperature dependent permeation of riboflavin. (Lee et al, Polymer, 1995, 36, 81)
Thin porous glass membranes were modified with vinyldimethylchlorosilane and then polymerization of N-isopropylacrylamide was effected by a redox system to produce composite membranes whose permeability increased above the LCST (Konno et al. in Polymer Gels Ed. D. DeRossi!, Plenum Press, New York, 1991, 173).
Similarly, the volume transition exhibited by NIPAAm in response to changes in temperature was used to control the pore size of beads grafted with NIPAAm in gel permeation chromatography and packed in chromatographic columns. For example, Gewehr et al. (Makromolekulare. Chemie, 1992, 193, 249) reacted carboxyl terminated PNIPAAm with amino groups functionalized porous glass beads and observed differences in exclusion limits of the composite upon changes in temperature. Hosoya et al. (Macromolecules, 1994, 27, 3973) used the process of U.S. Pat. No. 5,306,561 to produce polymer beads with a thermo-responsible surface layer that controls the access of compounds to the interior of the beads. However, since flow in a column packed with particles/beads occurs through the interstitial voids between packed particles, these grafted beads are not capable of forming thermal valves or thermal gates as in the present invention.
U.S. Pat. Nos. 5,334,310 and 5,453,185 disclose a process for preparing a continuous liquid chromatography column and a column containing a new class of macroporous polymer materials prepared by a simple molding process. The porous polymer monoliths are characterized by a unique bimodal pore distribution, consisting of large generally micrometer-sized convective pores and much smaller diffusive pores. High flow rates through the monoliths are obtained at low back pressures due to the network of the large canal-like convective pores which traverse the length of the monolith. It has now been discovered that unique flow-through properties can be imparted to these and other porous polymer materials by grafting the pores with thermally responsive polymer chains.
The concept of hydrophobic interaction chromatography (HIC) is based on the interaction of hydrophobic patches located on proteins with hydrophobic ligands located on a separation medium in an environment, such as an aqueous salt solution, that promotes these interactions. The column-bound ligands are typically short alkyl chains or phenyl groups. The strength of the interaction depends on many factors such as the intrinsic hydrophobicity of the protein, the type of ligands, their density, the separation temperature, and the salt concentration. Typically, the separation of two or more proteins is achieved by decreasing the salt concentration in the mobile phase, causing the less hydrophobic molecules to elute first. In contrast to highly hydrophobic reversed-phase chromatographic media that require elusion with organic solvents, the column surface incorporates a much lower density of ligands interspersed within a highly hydrophilic surface, allowing elusion with entirely aqueous eluents. The original column packings for hydrophobic interaction chromatography were derivatives of polysaccharide gels. Currently, a wide variety of hydrophobic media based on both inorganic and organic polymer beads is available for HIC. However, the re-equilibration of the column in the initial mobile phase after each analysis is a serious limitation for high throughput processes.
In prior isocratic hydrophobic interaction chromatography, a gradient solvent system with an increasing ionic strength was used to perform the elusions. In the present invention, however, a single solvent system at a single ionic strength, but at varying temperature, is used to perform the separation of two or more biomolecules, e.g. proteins, from each other.
Accordingly, it is an object of the present invention to provide thermally responsive polymer monoliths, particularly porous monoliths which are capable of functioning as thermal gates or thermal valves, more particularly porous monoliths suitable as a hydrophobic interaction chromatography substrate.
It is a further object to modify the internal pore surface of a polymer monolith with thermally responsive polymer chains.
It is a still further object to produce a polymer structure suitable for use in isocratic hydrophobic interaction chromatography wherein a single solvent is used to elute different proteins depending upon temperature.
These and still further objects will be apparent from the ensuing detailed disclosure of this invention.